Thiobacillus thiooxidans growth inhibitor

ABSTRACT

A Thiobacillus thiooxidans growth inhibitor containing as the active ingredient at least one substance selected from the group consisting of a tungsten powder and tungsten compounds. Under acidic conditions of pH value of 4 or less, this growth inhibitor sufficiently inhibits the growth of T. thiooxidans. When added in a very small amount to, for example, a structural material such as concrete in a sewage disposal plant, etc., it can therefore inhibit the growth of T. thiooxidans without substantially decreasing the strength, etc. of the structural material per se.

This application is a 371 of PCT/JP97/01323, filed on Apr. 17, 1997.

FIELD OF ART

The present invention relates to a Thiobacillus thiooxidans growthinhibitor, particularly relates to a growth inhibitor capable ofinhibiting growth of Thiobacillus thiooxidans in sewage-treatingfacilities to prevent deterioration of concrete structures or concretesewer pipes contacting sewage.

BACKGROUND OF THE INVENTION

It has been a problem that concrete structures in sewage-treatingfacilities become gypsiferous and deteriorated. This deterioration isknown to be ascribable to sulfur-oxidizing bacteria of the genusThiobacillus which oxidize hydrogen sulfide to sulfuric acid. Thesebacteria usually live widely in soil or water and grows by oxidation ofsulfur compounds and assimilation of carbon dioxide. Thesulfur-oxidizing bacteria of the genus Thiobacillus are known to existnot only in the portion of structures contacting sewage, but also instructures in the sewage-treating facility facing to the air but notcontacting sewage, depending on the species thereof.

Various methods have been proposed for preventing such deterioration ofthe structures. Most of practically performed methods include coatingthe structures with anti-corrosive materials. However, the coating ofthe anti-corrosive material can easily be damaged from pinholes orscratches, and thus has problem in durability. In addition, coating on ajoint of small-diameter tubes is difficult. Further, this method is notadvantageous in terms of cost.

Then, another method is proposed in Japanese Laid-open PatentApplication No.4-149053, in which deterioration of the structuralmaterials due to the sulfur-oxidizing bacteria of the genus Thiobacillusis prevented by directly admixing to concrete a particular metal or aparticular metal oxide insoluble in water but soluble in sulfuric acid,such as nickel, instead of coating the surface of the structures withthe anti-corrosive materials. In this method, nickel completely preventssulfur oxidation activity, respiration, and carbon dioxide gas-fixingactivity of the sulfur-oxidizing bacteria when pH value is aroundneutrality. This method is excellent for sufficiently preventingdeterioration due to the sulfur-oxidizing bacteria.

However, for practically inhibiting the growth of the sulfur-oxidizingbacteria to prevent deterioration of the concrete by adding nickel tothe concrete, about 0.1 parts by weight of nickel is being added to 100parts by weight of cement. It is therefore desired to develop aninhibitor that exhibits an excellent effect in inhibiting growth ofsulfur-oxidizing bacteria in smaller adding amount.

It is known that molybdenum, ammonium molybdate or a mixture of ammoniummolybdate and tungsten activates growth of Thiobacillus novellus,whereas tungsten, when used alone, inhibits growth of the same bacteria(Journal of Bacteriology, Vol.153, No.2 (1983) William M. et al."Sulfite Oxidase Activity in Thiobacillus novellus" p.941-944). On theother hand, it is also reported that, although molybdenum (Mo⁴⁺)activates growth of Thiobacillus novellus as noted above, it inhibitsgrowth of Thiobacillus thiooxidans (Chemical Abstracts, Vol. 95, No.1(Jul. 6, 1981) p127 (1081a)). Thus, even the sulfur-oxidizing bacteriaof the same genus Thiobacillus have different growth inhibitorymechanism. Therefore, even if a certain growth inhibitor is recognizedto have an effect on sulfur-oxidizing bacteria existing in neutral pHrange of 6 to 8 such as Thiobacillus novellus, such an inhibitor is notused in acidic pH range of 2 to 6, in which Thiobacillus novellusscarcely exists. Further, it is not believed that a substance whichinhibits growth of the aforementioned Thiobacillus novellus equallyinhibits growth of sulfur-oxidizing bacteria of the genus Thiobacillusexisting in pH range of 2 to 6 such as Thiobacillus thiooxidans.

Therefore, growth inhibitors proposed hitherto are not always effectiveto all of the sulfur-oxidizing bacteria of the genus Thiobacillus.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a Thiobacillusthiooxidans growth inhibitor that can sufficiently inhibit growth ofThiobacillus thiooxidans under the acidic condition at pH 4 or lower.

It is another object of the present invention to provide a Thiobacillusthiooxidans growth inhibitor that can inhibit Thiobacillus thiooxidansby admixing a very small amount of the inhibitor to a structuralmaterial such as concrete in a sewage-treating facility, and that doesnot substantially decrease strength of the structural material itself.

The present inventors proposed in Japanese Laid-open Patent ApplicationNo.4-149053 a method for preventing deterioration of a structuralmaterial due to the sulfur-oxidizing bacteria of the genus Thiobacillus.In the method, specific metals or specific metal oxides insoluble inwater but soluble in sulfuric acid, such as nickel, are directly admixedto concrete. As to Thiobacillus thiooxidans existing in neutral toacidic pH range of 2 to 6, sufficient growth inhibitory activity isobtained at the portion of the structural material directly contactingsewage to be kept in neutral pH range. However, the present inventorsfound out that inhibition of growth of the same Thiobacillus thiooxidansis reduced on the surface of the structural material near thesewage-treating facility but not in direct contact with the sewage underthe acidic condition at pH 4 or lower due to hydrogen sulfide in theair. It is not clear why the inhibition of the growth of the samebacteria varies depending on pH condition. However, it is assumed thatthe proposed nickel does not act directly on the bacteria themselves,but binds to an enzyme of the bacteria for exhibiting growth inhibitoryactivity, so that the binding to the enzyme is not sufficient under theacidic condition, resulting in insufficient growth inhibition. Thepresent inventors then searched for a substance which sufficientlyinhibits growth of Thiobacillus thiooxidans, which is ascribable to thedeterioration of the concrete under the acidic condition at pH 4 orlower, and found out that tungsten and tungsten compounds effectivelyinhibit growth of Thiobacillus thiooxidans existing under the acidiccondition, to thereby complete the present invention.

According to the present invention, there is provided a Thiobacillusthiooxidans growth inhibitor comprising at least one substance selectedfrom the group consisting of tungsten powders and a tungsten compound.

According to the present invention, there is also provided aThiobacillus thiooxidans growth inhibitor comprising at least onesubstance selected from the group consisting of nickel powders andnickel oxide powders, in addition to at least one substance selectedfrom the group consisting of tungsten powders and a tungsten compound.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the effect of Na₂ WO₄ on growth of NBI-3strain measured in Examples 1 to 3 and Comparative Example 1.

FIG. 2 is a graph showing the effect of Na₂ WO₄ on elemental sulfuroxidation activity of NBI-3 washed cells measured in Examples 4 to 7 andComparative Example 2.

FIG. 3 is a graph showing the effect of Na₂ WO₄ on elemental sulfuroxidation activity of NBI-3 washed cells measured in ComparativeExamples 3 to 7.

FIG. 4 is a graph showing the effect of Na₂ WO₄ on HSO₃ ⁻ oxygenactivity of a membrane protein of NBI-3 strain measured in Examples 8 to9 and Comparative Example 8.

FIG. 5 is a graph showing the effect of Na₂ WO₄ on a variety of cellsmeasured in Examples 10 to 14 and Comparative Examples 9 to 13.

FIG. 6 is a graph showing the effect on elemental sulfur oxidationactivity of NBI-3 washed cells measured in Examples 15 to 20 andComparative Examples 14 and 15.

FIG. 7 is a graph showing the effect on elemental sulfur oxidationactivity of NBI-3 washed cells measured in Examples 21 to 24.

PREFERRED EMBODIMENT OF THE INVENTION

The present invention will be described more in detail hereinbelow.

The present inhibitor inhibits growth of Thiobacillus thiooxidans, whichis known to be ascribable to deterioration of structural materials suchas concrete, mortar or high-molecular polymers in sewage-treatingfacilities. The present inhibitor can effectively inhibit the growth ofThiobacillus thiooxidans not only on a structural material that contactssewage directly but also on the surface of a structural material near asewage-treating facility under the acidic condition at pH 4 or lower dueto hydrogen sulfide in an air, to prevent the deterioration of thestructural material.

The present inhibitor contains as an active ingredient at least onesubstance selected from the group consisting of tungsten powders (W);and tungsten compounds such as W₂₈ O₅₈, Na₂ WO₄, CaWO₄, CaWO₄.H₂ O,CaWO₄.2H₂ O, or 5(NH₄)₂ O.12WO₃.nH₂ O (n=0 to 5). The active ingredientis assumed to bind to an enzyme of Thiobacillus thiooxidans under theacidic condition, particularly at pH 4 or lower, to inhibit sulfuroxidation, respiration and carbon dioxide gas-fixing of the bacteria, tothereby inhibit the growth of Thiobacillus thiooxidans. However, it isnot known how these active ingredients bind to the enzyme of thebacteria.

When the active ingredient of the present inhibitor is in the form ofpowders, the diameter thereof is not particularly limited. However, itis desirable that the diameter is in a range to enable expression of theeffective growth inhibition when the powders are admixed to thestructural material such as concrete and to enable distribution todesirable position in the structural material upon forming of thestructural material. Specifically, the diameter is desirably 0.005 to0.2 mm. If the diameter exceeds 0.2 mm, the growth inhibitory activitymay be reduced, thus being not preferable. If metal compounds soluble inwater or an organic solvent, such as Na₂ WO₄, are employed, these may beemployed in the form of a solution.

The effective concentration of the present inhibitor is preferably notless than 1 μmol/l, and more preferably 10 to 100 μmol/l with respect tothe growth environment for Thiobacillus Thiooxidans. For example, forpreventing the deterioration of the concrete and mortar due toThiobacillus thiooxidans, preferably 0.0001 to 0.1 parts by weight ofthe inhibitor may be added to 100 parts by weight of cement in theconcrete or mortar. Particularly, even by addition of small amount ofthe inhibitor, such as 0.0001 to 0.01 parts by weight, sufficient growthinhibition effect is obtained for a prolonged period of time, comparedto the conventional coating with anti-corrosive material.

The present inhibitor may further contain at least one substanceselected from the group consisting of nickel powders and nickel oxidepowders. With these substances contained, the inhibition of the growthof Thiobacillus thiooxidans in the pH range around neutrality can beimproved. There is no particular limitation to the diameter of thenickel powders and the nickel oxide powders. However, the diameter isdesirably 0.005 to 0.2 mm. The content of the nickel or nickel oxidepowders is desirably selected so that tungsten-containing substanceselected from the group consisting of tungsten powders and a tungstencompound: nickel powders and/or nickel oxide powders is in a range of0.1 to 1.0 by weight.

The present inhibitor may be admixed in the concrete by, e.g., adding adesirable amount of the inhibitor to a concrete composition, and moldingand curing the mixture in accordance with publicly known methods.Particularly, if the inhibitor material is in powder form, the materialmay easily and uniformly admixed in the concrete by centrifugalcompacting. The centrifugal compacting may be performed in accordancewith a publicly known method for producing Hume pipes. By suitablychoosing the condition for the centrifugal compacting, concrete havingdesired inhibitor may be produced. In the centrifugal compacting, thediameter of the inhibitor is important. If the diameter is too small,the inhibitor moves with bleeding water during the centrifugalcompacting and is possibly drained with the water. If the diameter istoo large, the inhibitor possibly moves radially outwards uponcentrifugal compacting. Thus, the diameter is preferably selectedsuitably from within the aforementioned preferable range.

The present inhibitor can sufficiently inhibit the growth ofThiobacillus thiooxidans even under the acidic condition at pH 4.5 orlower. By admixing very small amount of the present inhibitor to thestructural material such as concrete in sewage-treating facilities, thegrowth of Thiobacillus thiooxidans is sufficiently inhibited for a longperiod of time. Further, the present inhibitor does not substantiallydecrease strength of the structural material itself.

EXAMPLES OF THE INVENTION

The present invention will be explained in more detail with reference toExamples and Comparative examples, but the present invention is notlimited thereto.

Examples 1 to 3 and Comparative Example 1

20 ml of thiosulfate-inorganic salt medium (pH7.0) containing 0.2%sodium thiosulfate, 0.03% yeast extract, 0.3% (NH₄)₂ SO₄, 0.05% MgSO₄ -7H₂ O, 0.05% K₂ HPO₄, 0.01% KCl and 0.001% Ca(NO₃)₂.4H₂ O was inoculatedwith 1.0 g of corroded concrete, and kept under aerobic condition at 30°C. When pH of the medium decreased to 2.0, the medium was replaced by afresh medium. This culturing operation was repeated five times. A yellowcolony of Thiobacillus thiooxidans obtained by the culturing wasisolated, and this isolated strain was designated as NBI-3.

To 20 ml of elemental sulfur-inorganic salt medium (pH2.5) containing 1%elemental sulfur, 0.3% (NH₄)₂ SO₄, 0.05% MgSO₄.7H₂ O, 0.05% K₂ HPO₄,0.01% KCl and 0.001% Ca(NO₃)₂.4H₂ O was added an aqueous solutioncontaining 30 μmol/l of Na₂ WO₄ (Example 1), 300 μmol/l of Na₂ WO₄(Example 2) or 1000 μmol/l of Na₂ WO₄ (Example 3), or added nothing as acontrol (Comparative Example 1). With this mixture, NBI-3 strain wascultured. It is noted that Na₂ WO₄ is insoluble at pH 2.5. Propagationof NBI-3 strain with the lapse of time was measured by absorbance at 660nm. The results are shown in FIG. 1.

From the results of FIG. 1, it is seen that Na₂ WO₄ has an excellentThiobacillus thiooxidans growth inhibitory activity under acidiccondition at pH2.5.

Examples 4 to 7 and Comparative Example 2

To a reaction vessel of a Warburg manometer having a manometer, areaction vessel and a shaker were added 5 mg of washed cells of NBI-3cultured in Examples 1 to 3, 200 μl of β-alanine-SO₄ ²⁻ buffer (pH3.0)and 200 μmol of sodium sulfite, which were in total volume of 3 ml. Tothe vessel was further added an acqueous solution containing 10 μmol/lof Na₂ WO₄ (Example 4), 100 μmol/l of Na₂ WO₄ (Example 5), 1000 μmol/lof Na₂ WO₄ (Example 6) or 10000 μmol/l of Na₂ WO₄ (Example 7), or addednothing as a control (Comparative Example 2). Intake of oxygen by thecells with the lapse of time was measured, while pH in the reactionvessel was adjusted to 3.0 with 0.2 ml of sodium hydroxide charged inthe center well. The gas phase in the Warburg manometer was maintainedat 30° C. with air. The results are shown in FIG. 2.

Comparative Examples 3 and 4

Measurement was performed in the same manner as in Comparative Example 2except that boiled cells (Comparative Example 3) or non-washed cells(Comparative Example 4) were employed in place of the washed cells ofNBI-3 strain. The results are shown in FIG. 3.

Comparative Examples 5 to 7

Measurement was performed in the same manner as in Examples 4 to 7except that 1000 μmol/l of nickel of not larger than 200 mesh(Comparative Example 5), 10000 μmol/l of the same nickel (ComparativeExample 6) or 20000 μmol/l of the same nickel (Comparative Example 7)was employed in place of Na₂ WO₄. The results are shown in FIG. 3.

Comparing the results of FIG. 3 and FIG. 2, it is seen that theThicbacillus thiooxidans growth inhibitory effect of Na₂ WO₄ isextremely greater than that of nickel in the same amount.

Examples 8 and 9 and Comparative Example 8

To a reaction vessel of a Warburg manometer having a manometer, areaction vessel and a shaker were added 1 mg of membrane protein ofNBI-3 cultured in Examples 1 to 3, 200 μmol of MoPS/NaOH buffer (pH7.0)and 20 μmol of sodium sulfite. To the vessel was further added anaqueous solution containing 1000 μmol/l of Na₂ WO₄ (Example 8) or 10000μmol/l of Na₂ WO₄ (Example 9), or added nothing as a control(Comparative Example 8). Intake of oxygen by the cells with the lapse oftime was measured, while pH in the reaction vessel was adjusted to 7.0with 0.2 ml of sodium hydroxide charged in the center well. The gasphase in the Warburg manometer was maintained at 30° C. with air. Theresults are shown in FIG. 4.

Examples 10 to 14 and Comparative Examples 9 to 13

To 20 ml of elemental sulfur-inorganic salt medium (pH2.5) containing 1%elemental sulfur, 0.3% (NH₄)₂ SO₄, 0.05% MgSO₄.7H₂ O, 0.05% K₂ HPO₄,0.01% KCl and 0.001% Ca(NO₃)₂.4H₂ O was added an aqueous solutioncontaining 50 μmol/l of Na₂ WO₄, or added nothing as controls(Comparative Examples 9 to 13). To each of the mixtures was addedThiobacillus thiooxidans ON106 (Example 10), Thiobacillus thiooxidansON107 (Example 11), Thiobacillus thiooxidans IFO13701 (Example 12),Thiobacillus thiooxidans JCM3867 (Example 13) or NBI-3 strain (Example14), and cultured. Propagation of the cells of each strain was measuredby absorbance at 660 nm. The results are shown in FIG. 5.

From the results of FIG. 5, it is seen that Na₂ WO₄ has an excellentThiobacillus thiooxidans growth inhibitory activity under the acidiccondition at pH 2.5.

Examples 15 to 20 and Comparative Examples 14 and 15

To 20 ml of elemental sulfur-inorganic salt medium (pH2.5) containing 1%elemental sulfur, 0.3% (NH₄)₂ SO₄, 0.05% MgSO₄.7H₂ O, 0.05% K₂ HPO₄,0.01% KCl and 0.001% Ca(NO₃)₂.4H₂ O was added an aqueous solutioncontaining 100 μmol/l of tungsten (W) powders having a diameter of 10 to50μm (Example 15), 100 μmol/l of CaWO₄. 2H₂ O powders having a diameterof 10 to 25 μm (Example 16), 100 μmol/l of CaWO₄ powders having adiameter of 10 to 25 μm (Example 17), 100 μmol/l of W₂₈ O₅₈ powdershaving a diameter of 10 to 25 μm (Example 18), 100 μmol/l of 5(NH₄)₂O.12WO₃ powders having a diameter of 10 to 25 μm (Example 19), a mixtureof 100 μmol/l of Ni powders having a diameter of 10 to 50 and 100 μmol/lof W₂₈ O₅₈ powders having a diameter of 10 to 25 μm (Example 20) or 100μmol/l of Ni powders having a diameter of 10 to 50 μm (ComparativeExample 14), or added nothing as a control (Comparative Example 15).With this mixture, NBI-3 strain prepared in Example 1 was cultured.Propagation of NBI-3 strain with the lapse of time was measured byabsorbance at 660 nm. The results are shown in FIG. 6.

From the results of FIG. 6, it is seen that tungsten and varioustungsten compounds have an excellent Thiobacillus thiooxidans growthinhibitory activity under the acidic condition at pH2.5.

Example 21 to 24

To 20 ml of elemental sulfur-inorganic salt medium (pH7.0) containing 1%elemental sulfur, 0.3% (NH₄)₂ SO₄, 0.05% MgSO₄.7H₂ O, 0.05% K₂ HPO₄,0.01% KCl and 0.001% Ca(NO₃)₂.4H₂ O was added an aqueous solutioncontaining 100 μmol/l of W₂₈ O₅₈ powders having a diameter of 10 to 25μm (Example 21), a mixture of 100 μmol/l of Ni powders having a diameterof 10 to 50 and 100 μmol/l of W powders having a diameter of 10 to 50 μm(Example 22), a mixture of 100 μmol/l of Ni powders having a diameter of10 to 50 and 100 μmol/l of W₂₈ O₅₈ powders having a diameter of 10 to 25μm (Example 23) or a mixture of 100 μmol/l of Ni powders having adiameter of 10 to 50 and 100 μmol/l of 5(NH₄)₂ O.12WO₃ powders having adiameter of 10 to 25 μm (Example 24). With this mixture, NBI-3 strainprepared in Example 1 was cultured. Propagation of NBI-3 strain with thelapse of time was measured by absorbance at 660 nm. The results areshown in FIG. 7.

From the results of FIG. 7, it is seen that the inhibitor containingnickel exhibits an excellent Thiobacillus thiooxidans growth inhibitoryactivity at pH 7.0.

Examples 25 to 27

To 20 ml of elemental sulfur-inorganic salt medium (pH2.5) containing 1%elemental sulfur, 0.3% (NH₄)₂ SO₄, 0.05% MgSO₄.7H₂ O, 0.05K₂ HPO₄, 0.01%KCl and 0.001% Ca(NO₃)₂.4H₂ O was added an aqueous solution containing amixture of 100 μmol/l of Ni powders having a diameter of 10 to 50 and100 μmol/l of W powders having a diameter of 10 to 50 μm (Example 25), amixture of 100 μmol/l of Ni powders having a diameter of 10 to 50 and100 μmol/l of W₂₈ O₅₈ powders having a diameter of 10 to 25 μm (Example26) or a mixture of 100 μmol/l of Ni powders having a diameter of 10 to50 and 100 μmol/l of 5(NH₄)₂ O.12WO₃ powders having a diameter of 10 to25 μm (Example 27). With this mixture, NBI-3 strain prepared in Example1 was cultured. Propagation of NBI-3 strain with the lapse of time wasmeasured by absorbance at 660 nm. As a result, propagation of NBI-3strain was in zero level in any of Examples on the day 30.

What is claimed is:
 1. A composition comprising (1) a material selectedfrom the group consisting of concrete, mortar and high molecular weightpolymers, and (2) an effective amount of a growth inhibitor forThiobacillus thiooxidans selected from the group consisting of tungstenand tungsten compounds.
 2. The composition of claim 1, wherein thetungsten compound is selected from the group consisting of W₂₈ O₅₈, Na₂WO₄, CaWO₄, CaWO₄.H₂ O, CaWO₄.2H₂ O, 5(NH₄)₂ O.12WO₃.nH₂ O, where n is 0to 5, and mixtures thereof.
 3. The composition of claim 1 wherein thediameter of the tungsten or tungsten compound is about 0.005 to 0.2 mm.4. The composition of claim 1 wherein the concentration of the growthinhibitor is 0.0001 to 0.1 parts by weight based on 100 parts by weightof cement in said concrete or mortar.
 5. The composition of claim 1further comprising a nickel compound selected from the group consistingof nickel powders and nickel oxide powders.
 6. The composition of claim5 wherein the diameter of the nickel compound is about 0.005 to 0.2 mm.7. A method of inhibiting the growth of Thiobacillus thiooxidansbacteria, comprising contacting the bacteria with an effective amount oftungsten or a tungsten compound.
 8. The method of claim 7 wherein thetungsten compound is selected from the group consisting of W₂₈ O₅₈, Na₂WO₄, CaWO₄, CaWO₄.H₂ O, CaWO₄.2H₂ O, 5(NH₄)₂ O.12WO₃.nH₂ O, where n is 0to 5, and mixtures thereof.
 9. The method of claim 7 wherein thediameter of the tungsten or tungsten compound is 0.005 to 0.2 mm. 10.The method of claim 7 further comprising a nickel substance selectedfrom a group consisting of nickel powders, nickel oxide powders, andmixtures thereof.
 11. The method of claim 10 wherein the diameter of thenickel substance is 0.005 to 0.2 mm.
 12. The method of claim 7 whereinthe tungsten or tungsten compound is present in a structural materialselected from the group consisting of concrete, mortar and highmolecular weight polymers.
 13. The method of claim 7 wherein the growthof bacteria is at a pH of about 2 to
 6. 14. The method of claim 7,wherein the growth of bacteria is in a sewage treatment facility.